Not Applicable.
The complex structure of wood includes cellulose, hemicellulose and lignin, along with other minor components. Lignin is associated with cellulose and hemicellulose, and is probably partially covalently bound to both cellulose and hemicellulose. In the paper-making process, lignin is generally removed from the wood pulp since it lends a brownish color, reduces strength and imparts other undesirable characteristics to the finished product. Removal of lignin can be achieved in many ways.
A majority of the lignin is initially removed from wood pulp through chemical pulping (e.g., the Kraft process). In the subsequent bleaching process, chemical pulp is routinely reacted with chlorine and other delignifying chemicals to further remove lignin and then reacted with bleaching agents to modify the lignin from pulp, providing a stable brightened pulp. However, the treatment with chlorine is undesirable from an environmental standpoint because the resulting effluents contain a large number of toxic compounds (e.g,. chlorinated phenolics). Concern about the environmental harmful effects caused by pulp bleaching with chlorine containing chemicals has driven the industry to seek alternative bleaching methods.
Attempts to use xylanases and other enzymes derived from fungal and bacterial sources to enhance delignification and brightening, while lowering or eliminating the use of chlorine chemicals, have been described in the literature. However, existing enzyme systems generally do not readily achieve degradation of hemicellulose or delignification to a sufficient extent. The extent of hemicellulose degradation and delignification could be improved by employing additional xylanases that cleave xylan in a different manner or that act synergistically with other xylanases, hemicellulases, other enzymes, or even chemicals.
Numerous xylanases from fungal and bacterial microorganisms have been identified and characterized. (See, e.g., U.S. Pat. No. 5,437,992; Coughlin, M. P. supra; Biely, P. et al., Proceedings of the second TRICEL symposium on Trichoderma reesei Cellulases and Other Hydrolases, Espoo 1993, P. Souminen and T. Reinikainen eds., Foundation for Biotechnical and Industrial Fermentation Research 8:125-135 (1993)). In particular, three specific xylanases (XYL-I, XYL-II, and XYL-III) have been identified in T. reesei (Tenkanen, et al., Enzyme Microb. Technol. 14:566 (1992); Txc3x6rrxc3x6nen, et al., Bio/Technology 10:1461 (1992); and Xu, et al., Appl. Microbiol. Biotechnol. 49:718 (1998)).
Although numerous xylanases have been described in the literature, the need still exists to identify novel xylanases that are more effective in applications relating to animal feed, grain processing, biofuels, cleaning, fabric care, chemicals, plant processing, and delignifying and brightening of pulp and paper.
Applicants have identified cDNA clones that encode a novel xylanase referred to herein as XYL-IV and have certain sequence identity to previously-described xylanases.
In one embodiment, the invention provides an isolated nucleic acid molecule including DNA encoding XYL-IV. In certain aspects, the isolated nucleic acid includes DNA encoding an XYL-IV having the amino acid sequence of FIG. 2 (SEQ ID NO:2), or is complementary to such encoding nucleic acid sequences. Preferably, the DNA encoding a XYL-IV protein is derived from a microorganism, preferably a fungus or a bacterium. Preferably, the DNA is derived from a filamentous fungus such as Trichoderma spp., Humicola spp., Neurospora spp., Aspergillus spp., Fusarium spp., Penicillium spp., or Gliocladium spp., more preferably from Trichoderma spp. Also preferably, the DNA includes the nucleotide sequence of SEQ ID NO:1. Alternately, the DNA has at least 50%, 60%, or 70%, preferably at least 85% or 90%, sequence identity with the nucleotide sequence. of SEQ ID NO:1, or includes a derivative of the nucleotide sequence of SEQ ID NO:1, wherein the DNA encodes a XYL-IV protein which cleaves xylan, branched xylan or xylooligosaccharides. Vectors including such DNA, host cells having been transformed with such vectors and fermentation broths produced by such transformed host cells are also within the scope of the present invention.
Another embodiment of the present invention provides a partially or wholly isolated XYL-IV protein. Preferably, the XYL-IV is derived from a microorganism, preferably a fungus or a bacterium. Preferably, the XYL-IV is derived from a filamentous fungus, more preferably from a filamentous fungus such as Trichoderma spp., Humicola spp., Neurospora spp., Aspergillus spp., Fusarium spp., Penicillium spp., or Gliocladium spp., and most preferably from Trichoderma spp. In particular, the invention provides isolated native sequence XYL-IV, which in one embodiment, includes an amino acid sequence including residues 1 to 465 of FIG. 2 (SEQ ID NO:2). In a preferred embodiment of the present invention, the XYL-IV includes an amino acid sequence of SEQ ID NO:2, has at least 50% or 70%, preferably at least 85% or 90%, sequence identity with the amino acid sequence of SEQ ID NO:2, or includes a derivative of the amino acid sequence of SEQ ID NO:2, wherein the XYL-IV cleaves xylan.
Yet another embodiment of the present invention provides a method of producing XYL-IV protein including the steps of (a) obtaining a host cell which has been transformed with a vector including DNA encoding a XYL-IV protein; (b) culturing the host cell under conditions suitable for the expression and, optionally, secretion, of the XYL-IV protein; and (c) recovering the fermentation broth containing the XYL-IV protein.
In another embodiment, the invention provides an antibody which specifically binds to a XYL-IV protein or a domain thereof. Optionally, the antibody is a monoclonal antibody. In a still further embodiment, the invention provides methods using the XYL-IV protein or DNA encoding the XYL-IV protein.